Functional characterization of orbicularis oculi and extraocular muscles

The orbicularis oculi are the sphincter muscles of the eyelids and are involved in modulating facial expression. They differ from both limb and extraocular muscles (EOMs) in their histology and biochemistry. Weakness of the orbicularis oculi muscles is a feature of neuromuscular disorders affecting the neuromuscular junction, and weakness of facial muscles and ptosis have also been described in patients with mutations in the ryanodine receptor gene. Here, we investigate human orbicularis oculi muscles and find that they are functionally more similar to quadriceps than to EOMs in terms of excitation-contraction coupling components. In particular, they do not express the cardiac isoform of the dihydropyridine receptor, which we find to be highly expressed in EOMs where it is likely responsible for the large depolarization-induced calcium influx. We further show that human orbicularis oculi and EOMs express high levels of utrophin and low levels of dystrophin, whereas quadriceps express dystrophin and low levels of utrophin. The results of this study highlight the notion that myotubes obtained by explanting satellite cells from different muscles are not functionally identical and retain the physiological characteristics of their muscle of origin. Furthermore, our results indicate that sparing of facial and EOMs in patients with Duchenne muscular dystrophy is the result of the higher levels of utrophin expression

Longitudinal change of refraction over at least 5years in 15,000 patients

Background: To report the natural, longitudinal history of shifts in refractive errors in different age groups in a large western European cohort over at least 5years in the same patients. Methods: The electronic database of a large regional clinic containing 225,000 patients was searched for records of patients with a follow-up of at least 5years, excluding all patients who had received any surgical interventions in any eye. This search retrieved 15,799 patients aged 3months to 79years (median 37.8years) with refractive follow up of at least 5years (mean 8.8years) and no surgical interventions. Differences in spherical equivalents (sum of sphere +1/2 cylinder) and cylinder between first and last visit in the same patients in only the right eye were calculated, and used as the measure of refractive shift. Subsequently differences in change between the right and left eye were also determined. Results: Refractions were found to be mostly stable from 25 to 39years (n = 3,155 right eyes), with 50% of these patients not changing their refraction. In patients aged 20-24 (n = 825 right eyes), only 39% of the refractions remained stable, whereas 49% experienced a myopic shift. In the age group 40-69years (n = 6,694), 40-45% remained stable, with an increase in hyperopic shifts. Eighty-five percent of all patients had bilateral symmetric shifts, and 61% showed stable cylindrical values. Conclusions: This report documents clinical relevant changes in spherical equivalents in all age groups within 5 to 10years in the largest examined European cohort. Refractive surgery patients in particular should be selected accordingly, and be informed about the physiological changes which might still occur during their lifetim

Effects of daunorubicin, mitomycinC, azathioprine and cyclosporinA on human retinal pigmented epithelial, corneal endothelial and conjunctival cell lines

Background: We wished to investigate the toxicity of four immunosuppressant and antimetabolic drugs, which are known to influence postoperative wound healing, on three different human ocular cell lines. Methods: Acute toxicity to cyclosporinA, azathioprine, mitomicynC and daunorubicin was assessed in Chang cells by monitoring their uptake of propidium iodide during a 3-h period. Chronic toxicity was assessed by monitoring the proliferation and viability of subconfluent cultures of Chang cells, human corneal endothelial cells (HCECs) and retinal pigmented epithelial (RPE) cells after continuous exposure to the drugs for 7 days. Results: Acute toxicity testing revealed no obvious effects. However, the chronic toxicity tests disclosed a narrow concentration range over which cell proliferation decreased dramatically but calcein metabolism was sustained. Although the three lines reacted similarly to each agent, HCECs were the most vulnerable to daunorubicin and mitomycin. At a daunorubicin concentration of 0.05μg/ml, a 75% decrease in calcein metabolism (P<0.001) and a ≥95% cell loss (P<0.001) were observed. At a mitomycin concentration of 0.01μg/ml, cell density decreased by 61% (P<0.001) without a change in calcein metabolism, but at 0.1μg/ml, the latter parameter decreased to 12% (P=0.00014). At this concentration the proliferation of Chang and RPE cells decreased by more than 50%, whilst calcein metabolism was largely sustained. Cyclosporin inhibited cell proliferation moderately at lower concentrations (<5μg/ml; P=0.05) and substantially at higher ones, with a corresponding decline in calcein metabolism. Azathioprine induced a profound decrease in both parameters at concentrations above 5μg/ml. Conclusion: Daunorubicin, cyclosporin and azathioprine could be used to inhibit excessive intraocular scarring after glaucoma and vitreoretinal surgery without overly reducing cell viability. The attributes of immunosuppressants lie in their combined antiproliferative and immunomodulatory effect

Ocular penetration of caspofungin in a rabbit uveitis model

Background: Little is known about the ocular penetration of echinocandin antifungals. We studied the ocular distribution of systemically administered caspofungin in a rabbit uveitis model. Methods: Caspofungin (1mg/kg per day) was given intravenously to rabbits as a single dose or as repeated daily doses on 7days starting 24h after induction of unilateral uveitis by intravitreal endotoxin injection. Caspofungin concentrations were determined by high-performance liquid chromatography in the cornea, aqueous humor, vitreous humor, and serum 4, 8, 16, and 24h after administration of a single dose and 24h after the last of seven doses. Results: The mean caspofungin concentration in the aqueous of the inflamed eye 4 and 8h after single-dose administration was 1.30 ± 0.39μg/ml and 1.12 ± 0.34μg/ml, respectively. Drug concentrations decreased to 0.24 ± 0.09μg/ml at 16h and 0.26 ± 0.14μg/ml at 24h. In the vitreous of inflamed eyes drug levels were undetectable at all time points. No drug was found in the aqueous of inflamed eyes 24h after the last of seven repeated doses, and the vitreous only contained trace amounts. In the corneas of inflamed eyes concentrations reached 1.64 ± 0.48μg/g at 4h, peaked at 2.16 ± 1.14μg/g at 8h, and declined to 1.87 ± 0.52μg/g and 1.49 ± 0.48μg/g at 16and 24h, respectively. After repeated dosing, corneal concentrations of caspofungin were 0.8 and 1.0μg/g and below the limit of detection in two of four animals. In non-inflamed eyes no drug was detectable in the aqueous and vitreous humor, and the corneas at any time point. Conclusions: In our model, caspofungin reached therapeutically relevant levels in the aqueous and cornea but not in the vitreous humor of inflamed eyes. Intraocular drug deposition was critically dependent on a disrupted blood-eye barrier. These findings suggest a limited role for caspofungin in the treatment of fungal endophthalmiti

Tocotrienol inhibits proliferation of human Tenon's fibroblasts in vitro: a comparative study with vitamin E forms and mitomycin C

Purpose: To evaluate the potential of the vitamin E compound α-tocotrienol as antifibrotic agent in vitro. Methods: Using human Tenon's capsule fibroblast cultures, the antiproliferative and cytotoxic effects of the different vitamin E forms α-tocopherol, α-tocopheryl acetate, α-tocopheryl succinate and α-tocotrienol were compared with those of mitomycin C. To mimic subconjunctival and regular oral application in vivo, exposure time of serum-stimulated and serum-restimulated fibroblasts (SF and RF, respectively) to vitamin E forms was set at 6 days. Cultures were only exposed for 5min to mitomycin C due to its known acute toxicity and to mimic the short-time intraoperative administration. Proliferation (expressed as % of control) was determined by DNA content quantification on days 2, 4 and 6, whereas cytotoxicity was assessed by cell morphology and glucose 6-phosphate dehydrogenase (G6PD) release after 24h. Results: α-Tocopherol and α-tocopheryl acetate stimulated growth of SF, but not RF. Reduction of fibroblast content by α-tocopheryl succinate was accompanied by increased G6PD release and necrosis. Contrary to α-tocopheryl succinate, 50μM or repeatedly 20μM of α-tocotrienol significantly inhibited proliferation without causing cellular toxicity (maximal effect: 46.8%). RF were more sensitive to this effect than SF. Mitomycin C 100-400μg/ml showed a stronger antiproliferative effect than α-tocotrienol (maximal effect: 13.8%). Morphologic characteristics of apoptosis were more commonly found under treatment with mitomycin C. Conclusions: Of the vitamin E forms tested, only α-tocotrienol significantly inhibited growth at non-toxic concentrations. In this in vitro study, antiproliferative effects of mitomycin C were stronger than those of α-tocotrieno

Antifibrotic effects of tocotrienols on human Tenon's fibroblasts

Purpose: To compare the antifibrotic effect of vitamin E isoforms α-, γ-, and δ-tocotrienol on human Tenon's fibroblasts (hTf) to the antimetabolite mitomycin C. Methods: Antifibrotic effects of α- (40, 60, 80, 100, and 120μM), γ- (10, 20, 30, and 40μM) and δ-tocotrienol (10, 20, 30, and 40μM) on hTf cultures were evaluated by performing proliferation, migration and collagen synthesis assays. Whereas for vitamin E the exposure time was set to 7days to mimic subconjunctival application, cultures were exposed only 5 min to mitomycin C 100μg/ml to mimic intraoperative administration. Cell morphology (phase contrast microscopy) as an assessment for cytotoxicity and cell density by measuring DNA content in a fluorometric assay to determine proliferation inhibition was performed on day 0, 4, and 7. Migration ability and collagen synthesis of fibroblasts were measured. Results: All tested tocotrienol isoforms were able to significantly inhibit hTf proliferation in a dose-dependent manner (maximal inhibitory effect without relevant morphological changes at day 4 for α-tocotrienol 80μM with 36.7% and at day 7 for α-tocotrienol 80μM with 42.6% compared to control). Degenerative cell changes were observed in cultures with concentrations above 80μM for α- and above 30μM for γ- and δ-tocotrienol. The highest collagen synthesis inhibition has been found with 80 µM α-tocotrienol (62.4%) and no significant inhibition for mitomycin C (2.5%). Migration ability was significantly reduced in cultures exposed to 80 µM α- and 30 µM γ-tocotrienol (inhibition of 82.2% and 79.5%, respectively, compared to control) and also after mitomycin C treatment (60.0%). Complete growth inhibition without significant degenerative cell changes could only be achieved with mitomycin C. Conclusion: In vitro, all tested tocotrienol isoforms were able to inhibit proliferation, migration and collagen synthesis of human Tenon's fibroblasts and therefore may have the potential as an anti-scarring agent in filtrating glaucoma surger

Fate of the esophagogastric anastomosis

ObjectiveThe study objective was to evaluate histopathology of the esophagogastric anastomosis after esophagectomy, determine time trends of histologic changes, and identify factors influencing those findings.MethodsA total of 231 patients underwent 468 upper gastrointestinal endoscopies with anastomotic biopsy a median of 3.5 years after esophagectomy. Mean age was 59 ± 12 years, 74% (171) were male, and 96% (222) were white. Seventy-eight percent (179) had esophagectomy for cancer, 13% (30) had chemoradiotherapy, and 13% (30) had prior esophageal surgery. The anastomosis was 20 ± 2.0 cm from the incisors. Anti-reflux medications were used in 59% of patients (276/468) at esophagoscopy. Histopathology was graded as normal (0), consistent with reflux (1), cardia mucosa (2), intestinal metaplasia (3), and dysplasia (4). Repeated-measures nonlinear time-trend analysis and multivariable analyses were used.ResultsGrades 0 and 1 were constant, 5% and 92% at 10 years, respectively. Anti-reflux medication, induction therapy, and higher anastomosis were predictive of less grade 1 histopathology. Grades 2 and 3 increased with time: 12% and 33% at 5 years and 4% and 16% at 10 years, respectively. No variable was predictive of grade 2 or 3 (P > .15) except passage of time. No patient’s condition progressed to dysplasia or cancer.ConclusionsThe esophagogastric anastomosis is subject to gastroesophageal reflux. To minimize histopathologic changes of reflux, the anastomosis should be constructed as high as possible (closer to incisors) and anti-reflux medications prescribed. Surveillance endoscopy, if performed, will document a time-related progression of reflux-related histopathologic changes. However, during surveillance, intestinal metaplasia is uncommon and progression to cancer rare

Quantifying Forest Structure Within Two Managed Units in Rock Cut State Park

The 1251 ha Rock Cut State Park (RCSP) is the largest state park in northern Illinois . Over the past 56 years, management practices have included prescribed burning, regular mowing, and tree plantings . Specifically, we sought to quantify the species composition, age structure, and light environment in two management units within the park . The first unit (campground) was last burned in 2009 and is comprised of primarily small trees; the second unit (Willow Creek Natural Area – WCNA) is more open and relatively undisturbed with larger trees . To assess these two units we used several methods, including stand structure analysis, tree coring, and hemispherical photographs . Between the months of June-August (2012 and 2013), eight plots were delineated and sampled . Within the campground unit, median tree age was 52 .5 years, with a maximum age of approximately 130 years . This site is mostly dominated by young slippery elm (Ulmus rubra) followed by white oak (Quercus alba) and hickory (Carya spp .) . Elimination of landscape-scale fires has likely led to a conversion from oak savanna to the closed forest found today at RCSP . High densities of shade-tolerant species dominating WCNA suggest management fires have been insuff icient at maintaining white and bur oak regeneratio